Jessica A. Sorrentino

Monitoring tumorigenesis and senescence in vivo with a p16 INK4a-luciferase model

Monitoring cancer and aging in vivo remains experimentally challenging. Here, we describe a luciferase knockin mouse (p16(LUC)), which faithfully reports expression of p16(INK4a), a tumor suppressor and aging biomarker. Lifelong assessment of luminescence in p16(+/LUC) mice revealed an exponential increase with aging, which was highly variable in a cohort of contemporaneously housed, syngeneic mice. Expression of p16(INK4a) with aging did not predict cancer development, suggesting that the accumulation of senescent cells is not a principal determinant of cancer-related death. In 14 of 14 tested tumor models, expression of p16(LUC) was focally activated by early neoplastic events, enabling visualization of tumors with sensitivity exceeding other imaging modalities. Activation of p16(INK4a) was noted in the emerging neoplasm and surrounding stromal cells. This work suggests that p16(INK4a) activation is a characteristic of all emerging cancers, making the p16(LUC) allele a sensitive, unbiased reporter of neoplastic transformation.

Effect of Cytotoxic Chemotherapy on Markers of Molecular Age in Patients With Breast Cancer

BACKGROUND Senescent cells, which express p16 (INK4a) , accumulate with aging and contribute to age-related pathology. To understand whether cytotoxic agents promote molecular aging, we measured expression of p16 (INK4a) and other senescence markers in breast cancer patients treated with adjuvant chemotherapy. METHODS Blood and clinical information were prospectively obtained from 33 women with stage I to III breast cancer at four time points: before anthracycline-based chemotherapy, immediately after anthracycline-based chemotherapy, 3 months after anthracycline-based chemotherapy, and 12 months after anthracycline-based chemotherapy. Expression of senescence markers p16 (INK4a) and ARF mRNA was determined using TaqMan quantitative reverse-transcription polymerase chain reaction in CD3(+) T lymphocytes, telomere length was determined by Southern analysis, and senescence-associated cytokines were determined by enzyme-linked immunosorbent assay. Findings were independently assessed in a cross-sectional cohort of 176 breast cancer survivors enrolled a median of 3.4 years after treatment; 39% previously received chemotherapy. All statistical tests were two-sided. RESULTS In prospectively analyzed patients, expression of p16 (INK4a) and ARF increased immediately after chemotherapy and remained elevated 12 months after treatment. Median increase in log2 p16 (INK4a) was 0.81 (interquartile range = 0.28-1.62; Wilcoxon signed-rank P < .001), or a 75% absolute increase in expression, equivalent to the increase observed over 14.7 years of chronological aging. ARF expression was comparably increased (P < .001). Increased expression of p16 (INK4a) and ARF was associated with dose-dense therapy and hematological toxicity. Expression of two senescence-associated cytokines (VEGFA and MCP1) was durably increased by adjuvant chemotherapy. Telomere length was not affected by chemotherapy. In a cross-sectional cohort, prior chemotherapy exposure was independently associated with a log2-increase in p16 (INK4a) expression of 0.57 (repeated measures model, P < .001), comparable with 10.4 years of chronological aging. CONCLUSIONS Adjuvant chemotherapy for breast cancer is gerontogenic, inducing cellular senescence in vivo, thereby accelerating molecular aging of hematopoietic tissues.

p16INK4a reporter mice reveal age-promoting effects of environmental toxicants

While murine-based systems to identify cancer-promoting agents (carcinogens) are established, models to identify compounds that promote aging (gerontogens) have not been described. For this purpose, we exploited the transcription of p16INK4a, which rises dynamically with aging and correlates with age-associated disease. Activation of p16INK4a was visualized in vivo using a murine strain that harbors a knockin of the luciferase gene into the Cdkn2a locus (p16LUC mice). We exposed p16LUC mice to candidate gerontogens, including arsenic, high-fat diet, UV light, and cigarette smoke and serially imaged animals to monitor senescence induction. We show that exposure to a high-fat diet did not accelerate p16INK4a expression, whereas arsenic modestly augmented, and cigarette smoke and UV light potently augmented, activation of p16INK4a-mediated senescence. This work provides a toxicological platform to study mammalian aging and suggests agents that directly damage DNA promote molecular aging.

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

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

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

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

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

The concomitant use of CDK4/6 inhibitors with cytotoxic agents may prevent chemotherapy-induced bone marrow exhaustion in cancer patients. Rock-a-bye stem cells Traditional chemotherapy can be quite effective against cancer, but its efficacy is often reduced by dose-limiting side effects. Because this type of therapy kills rapidly dividing cells, one of the most common toxicities is hematologic, resulting from chemotherapy drugs killing normal hematopoietic cells in the bone marrow. To protect the healthy hematopoietic stem cells, He et al. devised a method of combining chemotherapy with an inhibitor of cyclin-dependent kinases 4 and 6, which transiently suppresses the cell cycle in normal cells but not in malignant ones. The authors tested their drug with chemotherapy in mice to show protection from myelotoxicity, and they performed a human trial demonstrating its safety in healthy volunteers. Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise (“exhaustion”), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer.

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

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

Abstract 1784: Pre-clinical characterization of G1T28-1, a novel CDK 4/6 inhibitor for protection of bone marrow from cytotoxic chemotherapies

G1T28-1 is a clinical stage, small molecule inhibitor of cyclin dependent kinases 4/6 (CDK4/6). Hematopoietic stem and progenitor cells (HSPC) require CDK4/6 for proliferation, and CDK4/6 inhibition allows the transient arrest of HSPC in the G1 phase of the cell cycle. This arrest may reduce the sensitivity of HSPC to DNA damaging chemotherapies by limiting G1 to S-phase progression in the setting of unrepaired DNA damage. Reducing HSPC death may reduce chemotherapy-induced myelosuppression (CIM), the major dose-limiting toxicity of most cytotoxic anti-cancer agents. G1T28-1 was specifically designed with high potency, exquisite selectivity, and favorable pharmacology to induce a predictable and well-defined transient arrest of HSPC as compared to less potent and selective CDK4/6 inhibitors. Biochemical profiling demonstrates that G1T28-1 is a competitive inhibitor of CDK4/6 at low nanomolar concentrations, and that G1T28-1 is highly selective for CDK4/cyclin D1 and CDK6/cyclin D3 as compared to CDK2/cyclin A or CDK2/cyclin E. G1T28-1 induces a clean G0/G1 arrest in CDK4/6 dependent cell lines in vitro. Since the down-stream target of CDK4/6 is the retinoblastoma protein (Rb), we investigated the ability of G1T28-1 to inhibit Rb phosphorylation. In CDK4/6 dependent cell lines, G1T28-1 exposure fully blocks Rb phosphorylation by 16 hours, while no effect on Rb in CDK4/6-independent cells is observed. To determine the duration and reversibility of G1T28-19s effects on cells in culture, Rb competent cells were treated with G1T28-1 for 24 hours, and then the drug was washed out. While cells were arrested at 24 hours, they re-entered the cell cycle by 16 hours after washout and maintained normal cell cycle kinetics thereafter. To demonstrate that the G0/G1 arrest induced by G1T28-1 decreases DNA damage and apoptosis following exposure to chemotherapeutic agents, Rb competent cells were pre-treated with G1T28-1 or vehicle control for 16 hours followed by incubation with various chemotherapies. Cells were then assayed for γ-H2AX formation and caspase activation. Treatment with G1T28-1 prior to DNA damaging agents attenuates DNA damage as measured by γ-H2AX formation and decreased caspase 3/7 activation in a dose-dependent manner, indicating a decrease in chemotherapy-induced apoptosis. In Rb null cancers, such as small cell lung cancer (SCLC), G1T28-1 may effectively protect HSPCs without affecting the chemotherapy9s efficacy. In vitro testing with SCLC cell lines confirmed that chemotherapy efficacy was unaffected by the addition of G1T28-1. In summary, G1T28-1 is a novel potent and selective CDK4/6 inhibitor that induces a transient and reversible G0/G1 cell cycle arrest in CDK4/6-sensitive cells CDK4/6-dependent cells, thereby decreasing CIM. However, the anti-tumor activity of chemotherapy in CDK4/6-resistant cells is unaffected by G1T28-1. Citation Format: John E. Bisi, Hannah S. White, Jessica A. Sorrentino, Patrick J. Roberts, Jay C. Strum. Pre-clinical characterization of G1T28-1, a novel CDK 4/6 inhibitor for protection of bone marrow from cytotoxic chemotherapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2015-1784

Abstract 5641: Effects of G1T48, a novel orally bioavailable selective estrogen receptor degrader (SERD), and the CDK4/6 inhibitor, G1T38, on tumor growth in animal models of endocrine resistant breast cancer

Background: The combination of targeting the CDK4/6 and ER signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here we report the preclinical characterization of G1T48, a novel, orally bioavailable, non-steroidal small molecule inhibitor of ERα, which is a potent, selective antagonist that down regulates ERα in vitro and in vivo in ER-positive models of breast cancer. Methods: Breast cancer cells expressing clinically relevant ESR1 mutations (ER-Y537S, ER-D538G) were threated with G1T38, G1T48, and mechanistically distinct SERMs/SERDs and cellular proliferation was assessed by measuring DNA content (Hoechst dye). Ovariectomized nu/nu mice bearing xenograft tumors of clinically relevant tamoxifen (TamR) and aromatase (long term estrogen deprived) resistant ER+ breast cancer were treated with G1T38 and G1T48, alone or in combination, with clinically relevant comparators. Time to progression and tumor volume were assessed over a 4 week dosing period. Results: G1T38 and G1T48 significantly inhibited cellular proliferation of MCF7 breast cancer cells bearing endocrine resistant ER mutations, ER-Y537S and ER-D538G. G1T48 treatment led to dramatic reductions in ER protein levels. Importantly, tumor growth inhibition was observed in mouse models of sensitive and resistant human breast cancer when G1T48 was dosed as a single agent or in combination with G1T38, a potent, selective CDK4/6 inhibitor. Conclusions: G1T38, a novel CDK4/6 inhibitor, and G1T48, a novel SERD, either alone or in combination, demonstrated highly potent inhibition of tumor growth in animal models of tamoxifen and aromatase resistance. G1T48 also demonstrated activity in models of endocrine resistance mediated by ER mutation. G1T48 is currently completing IND enabling studies. Citation Format: Suzanne E. Wardell, Alexander P. Yllanes, Jennifer G. Baker, Robert M. Baldi, Taylor K. Krebs, Jessica Sorrentino, John Bisi, Jay Strum, John D. Norris. Effects of G1T48, a novel orally bioavailable selective estrogen receptor degrader (SERD), and the CDK4/6 inhibitor, G1T38, on tumor growth in animal models of endocrine resistant breast cancer [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 5641. doi:10.1158/1538-7445.AM2017-5641

Abstract 2824: G1T38, a novel, oral, potent and selective CDK4/6 inhibitor for the treatment of Rb competent tumors

The development of therapeutically effective inhibitors of the cyclin dependent kinase (CDK) family has been challenging due to a poor understanding of target and structural biology leading to the development of drugs that are toxic with limited efficacy. Recently, the first highly selective CDK4/6 inhibitor, palbociclib, was approved by the FDA for use in combination with letrozole as a first line treatment in patients with ER+/ HER2- metastatic breast cancer. While this first approved CDK4/6 inhibitor is highly efficacious, it causes severe myelosuppression during daily treatment resulting in at least a 7 day treatment holiday in every 28 day cycle to allow recovery of neutrophil counts. This leads to an increased risk of febrile neutropenia, potential tumor growth during the treatment holiday, and emergence of drug resistance. CDK4/6-induced myelosuppression is the result of on-target inhibition of hematopoietic stem and progenitor cell proliferation causing a narrow therapeutic window between tumor efficacy and neutropenia. Thus, a next generation CDK4/6 inhibitor will need to produce a potent, selective G1 arrest in Rb competent tumors while minimizing the effect on the bone marrow. With a new understanding of CDK4/6 biology, we have generated compounds with unique properties that maintain tumor efficacy while minimizing inhibition of bone marrow proliferation. Here we describe G1T38, a novel, oral, potent, and selective CDK4/6 inhibitor. Biochemical profiling demonstrates G1T38 is a competitive, nanomolar inhibitor of CDK4/6 with highly selectivity for CDK4-cyclin D1 and CDK6-cyclin D3. Kinome profiling exhibits on-target selectivity across 468 independent kinases at 100 nM, with no significant activity against other cell cycle or mitotic kinases. G1T38 elicits a precise G1 arrest profile along with loss of Rb phosphorylation in Rb competent cells with no impact in Rb deficient cells up to three orders of magnitude above the biochemical IC50. In cell proliferation assays, G1T38 exhibits a low EC50 ( 3 μM in Rb null cells. In vivo, daily oral treatment with G1T38 causes significant, durable growth inhibition of tumors in a HER2/neu GEMM and in MCF7 xenograft breast cancer models. G1T38 is cleared from the plasma but significantly accumulates in tumors. The level of G1T38 in the tumor correlates with significant reductions in Rb phosphorylation and tumor cell proliferation. Additionally, daily oral dosing of G1T38 in mice, rats and dogs for up to 28 days has shown a dose dependent decrease in neutrophils without severe neutropenia. These data demonstrate that the unique pharmacokinetic and pharmacodynamic properties of G1T38 allow it to be highly efficacious against tumors while having a mild effect on the bone marrow, thus making it optimal for use as a daily oral antineoplastic agent. Citation Format: Jessica A. Sorrentino, John E. Bisi, Patrick J. Roberts, Jay C. Strum. G1T38, a novel, oral, potent and selective CDK4/6 inhibitor for the treatment of Rb competent tumors. [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 2824.