John Bisi - Academia.edu (original) (raw)

Papers by John Bisi

G1T28 shows a dose-dependent decrease in cycling bone marrow cells in beagle dogs with minimal ch... more G1T28 shows a dose-dependent decrease in cycling bone marrow cells in beagle dogs with minimal changes in peripheral blood neutrophils and RBCs after one G1T28 treatment.

Molecular and Cellular Biology / Genetics, Jul 1, 2019

The family of cyclin-dependent kinases (CDKs) are serine/threonine protein kinases important for ... more The family of cyclin-dependent kinases (CDKs) are serine/threonine protein kinases important for cell cycle control and/or regulation of transcription. CDK9/CyclinT1 is a key regulator of transcription in eukaryotic cells and has been shown to be dysregulated at the level of protein and kinase activity in both hematologic and solid tumors. CDK9/CyclinT1 forms the active P-TEFb complex and phosphorylates Ser2 residues in the carboxy-terminal domain of RNA polymerase II (RNApol II) to initiate elongation of mRNA transcripts. CDK9 activity regulates transcription of a variety of short-lived transcripts that promote survival and directly suppress apoptosis in cancer cells, including MYC,XRN2, MCL-1,and XIAP. MYC-driven tumor types with Rb-loss or high expression levels of cyclin E, such as triple negative breast cancer (TNBC), are difficult to treat and are resistant to existing CDK4/6 inhibitors. Since CDK9 is upstream of these oncogenic drivers, inhibition of CDK9 could potentially bypass innate resistance mechanisms and induce cell death in TNBC through blocking expression of MYC and MCL-1, for example. To target CDK9 in TNBC, we used structure-based drug design and developed a library of novel, potent and selective CDK9 inhibitors (CDK9i). The lead CDK9i exhibit single digit nanomolar potency against CDK9/CyclinT1 complexes and good selectivity against other CDK family members in biochemical assays. In addition, they also display high selectivity over 502 other kinases. Lead CDK9 inhibitors were found to potently inhibit phosphorylation of RNApol II Ser2 in a time and dose dependent manner in TNBC cells and decrease MYC, MCL-1 and XIAP at both mRNA and protein levels. Additionally, treatment of TNBC cells led to a potent G2/M cell cycle arrest, inhibition of cell proliferation and induction of apoptosis within 24 hours, regardless of Rb status of the tumor cells. Primary human bone marrow and normal human fibroblast cells, however, did not undergo apoptosis within 72 hours of treatment with CDK9i, suggesting a potential therapeutic window for CDK9i treatment. Further studies are ongoing to assess the effect of dose and scheduling on tumor efficacy in mouse TNBC tumor models. Selective inhibition of CDK9 presents a novel treatment strategy for difficult-to-treat tumor types, including those resistant to existing targeted therapeutics. Citation Format: Hailey E. Brighton, Claire R. Hall, Kerry A. Dillon, John E. Bisi, Jay C. Strum. Selective inhibition of CDK9 induces apoptosis of TNBC cells independent of Rb status [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 4426.

The development of therapeutically effective inhibitors of the cyclin dependent kinase (CDK) fami... more 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 (<100 nM) in Rb competent cell lines compared to >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.

G1T28-1 is a clinical stage, small molecule inhibitor of cyclin dependent kinases 4/6 (CDK4/6). H... more 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 G1T28-1's 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 chemotherapy's 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

Science, Nov 27, 1992

Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing d... more Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 1 5-mer PNA to the transcribed strand of a G-free transcription cassette caused 90to 100 percent site-specific termination of pol 11 transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA. RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in ,B-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017

Inhibition of the p16 INK4a /cyclin D/CDK4/6/RB pathway is an effective therapeutic strategy for ... more Inhibition of the p16 INK4a /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.

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine... more Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017

Conventional cytotoxic chemotherapy is highly effective in certain cancers, but causes doselimiti... more Conventional cytotoxic chemotherapy is highly effective in certain cancers, but causes doselimiting 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. Here, we show that the co-administration of G1T28 (trilaciclib), a small-molecule inhibitor of cyclindependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects

Cancer Research, Jul 1, 2019

The cyclin dependent kinase (CDK) family of proteins is associated with cell cycle progression an... more The cyclin dependent kinase (CDK) family of proteins is associated with cell cycle progression and transcriptional regulation. Abnormalities in the cell cycle, including modifications to the function of CDKs, their regulators (cyclins), or their natural inhibitors, are frequently associated with the formation and growth of tumors. While recent advances in treatments using CDK inhibition have focused on targeting CDK4/6, with regulatory approvals of palbociclib, ribociclib, and abemaciclib, these compounds only target tumors sensitive to CDK4/6 inhibitors. We are focused on developing a novel, potent, and selective inhibitor of CDK2 to treat patients whose tumors are insensitive to CDK4/6 inhibition, either by primary resistance or acquired resistance by prior treatment with a CDK4/6 inhibitor. CDK2, a serine-threonine kinase, is a member of the CDK family that binds two regulatory cyclins, E and A. CDK2 when complexed to cyclin E is involved in the G1 to S-phase transition. However, when CDK2 is complexed with cyclin A, the cell will progress through the S to M-phase. Overexpression of cyclin E has been described in a subset of triple negative breast cancer (TNBC), tumors with acquired resistance to CDK4/6 inhibition such as ER+ Her2- breast cancer, as well as ovarian, lung, and other tumor types. We have utilized medicinal chemistry and structure activity relationship (SAR) modeling, starting from our proprietary scaffold, to generate a series of small molecule CDK2 inhibitors with drug-like properties. These compounds were initially screened for potency and selectivity using an array of biochemical and in-vitro assays. We identified multiple small molecule inhibitors with sub-nanomolar biochemical IC50s for CDK2 when complexed with either cyclin E or cyclin A and evaluated their activity in normal cell lines, a breast cancer cell line resistant to CDK4/6 inhibition, TNBC, and other tumor cell lines. We examined cell cycle analysis, EdU incorporation, cell proliferation, caspase activation, and western blot analysis of genes associated with the downstream targets of CDK2. These CDK2 inhibitors potently arrest cells in the G2/M-phase and inhibit proliferation in a manner dependent on time and dose, with a corresponding decrease in phosphorylated Rb. Our lead compound was also evaluated in mouse xenograft studies for efficacy, investigating tumor growth inhibition in a model with cyclin E overexpression and a model with an acquired resistance to CDK4/6 inhibition. These potent CDK2 inhibitors demonstrate a potentially promising method of treating tumors with primary or acquired resistance to CDK4/6 inhibitors. Citation Format: Claire R. Hall, John E. Bisi, Jay C. Strum. Inhibition of CDK2 overcomes primary and acquired resistance to CDK4/6 inhibitors [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 4414.

Journal of Clinical Oncology, May 20, 2016

e20099Background: Small cell lung cancer (SCLC) is an aggressive form of lung cancer characterize... more e20099Background: Small cell lung cancer (SCLC) is an aggressive form of lung cancer characterized by loss of the tumor suppressor Rb. Chemotherapy and radiation remain the standard of care for SCL...

The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cy... more The recent FDA approvals of palbociclib, ribociclib, and abemaciclib in breast cancer validate cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets, and warrant investigations in other tumor types with frequently observed alterations in the CDK4/6 pathway. Towards this end, we assessed the preclinical antitumor efficacy of G1T38, an oral, potent, and selective small-molecule CDK4/6 inhibitor in clinical development, in models of non-small cell lung cancer (NSCLC). Importantly, preclinical studies have shown that G1T38 has a differentiated and potential best-in-class profile since it can be dosed continuously without causing severe neutropenia, which could result in better tumor efficacy. Initial efficacy screening of G1T38 in 60 patient-derived NSCLC xenografts revealed significant tumor growth inhibition (TGI), primarily in lung adenocarcinomas harboring frequently observed oncogenic alterations in KRAS, EGFR, BRAF, and ALK. In vitro analyses show that CDK4/6 inhibition enhances the antiproliferative effect of inhibitors targeting these oncogenes (or their signaling pathways), suggesting a possible role for G1T38 in augmenting response and/or delaying acquired resistance to agents currently utilized in the clinic. These results were validated in vivo in mouse NSCLC xenograft models with defined oncogenic alterations, including EGFR mutations. For example, in H1975 xenografts harboring EGFR L858R/T790M (the T790M mutation is responsible for over 50% of cases of acquired resistance to first-generation EGFR inhibitors), combination of G1T38 with erlotinib resulted in 77% TGI after 18 days–a significant improvement over either therapy alone. Moreover, in H1975 xenografts, G1T38 delayed resistance to the second-generation EGFR inhibitor afatinib, and upon development of resistance to afatinib monotherapy, addition of G1T38 to afatanib treatment resulted in stabilization of tumor growth. G1T38 also augmented the response of EGFR-mutant NSCLC xenografts to the third-generation EGFR inhibitor osimertinib, significantly enhancing TGI compared to either therapy alone. As a result, a safety, pharmacokinetics, and efficacy study of G1T38 + osimertinib (G1T38-03) is scheduled for initiation in 1Q18 in patients with EGFR-mutant, T790M-positive NSCLC. Additional efforts are ongoing to investigate the combination of G1T38 with targeted therapies in NSCLC murine models harboring alterations in KRAS, ALK, and BRAF. Together, our results suggest a compelling rationale for utilizing G1T38 as a backbone for multiple targeted therapy combination regimens in NSCLC. Citation Format: Jessica A. Sorrentino, Daniel M. Freed, John E. Bisi, Jay C. Strum, Patrick J. Roberts. The CDK4/6 inhibitor G1T38 enhances response to targeted therapies in preclinical models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1522.

Cancer Research

Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with ... more Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with regulatory subunits called cyclins to drive cell cycle progression. Uncontrolled cellular proliferation is a hallmark of cancer commonly driven by dysregulated kinase activity of specific CDK family members, including cyclin-dependent kinase 2 (CDK2). Aberrant CDK2 activity most frequently occurs through amplification of CCNE1 and/or overexpression of its protein product cyclin E1, which is a canonical binding partner of CDK2. Overexpression of cyclin E1 is observed in many solid tumors including in patients with high grade serous ovarian cancer (HGSOC), gastric cancer, and ER-positive breast cancer patients whose tumors have progressed on a prior CDK4/6 inhibitor regimen. Selective inhibition of CDK2 is thus a compelling therapeutic approach to regain cell cycle control. Here, we report preclinical data supporting the development of INX-315 for patients with cancers in which proliferati...

Target Genes with greater than 90% inhibition at 100nM

Cyclin-dependent kinases (CDKs) mediate cell cycle progression, regulating transition from G1 to ... more Cyclin-dependent kinases (CDKs) mediate cell cycle progression, regulating transition from G1 to S phase and G2 to M phase. CDK activity is tightly controlled throughout the cell cycle by posttran-scriptional modifications as well as the expression of cyclins and CDK inhibitors. There are four proliferative CDKs: CDK1, which predominantly regulates the transition from G2 to M phase, and CDK2/4/6, which regulate the transition from G1 to S phase (1,2). Recent work has shown that there is considerable redun-dancy among the CDKs that regulate the transition from the G1 to S phase, and many proliferating cells can use any of these

L'invention concerne des composes et des procedes ameliores permettant de traiter des cancers... more L'invention concerne des composes et des procedes ameliores permettant de traiter des cancers et des troubles hyper-proliferatifs selectionnes.

Molecular and Cellular Biology / Genetics, 2019

Molecular and Cellular Biology / Genetics, Jul 1, 2019

Science, Nov 27, 1992

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017

Cancer Research, Jul 1, 2019

Journal of Clinical Oncology, May 20, 2016

Cancer Research

Target Genes with greater than 90% inhibition at 100nM

Molecular and Cellular Biology / Genetics, 2019