Abstract Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of cyclin-dependent kinase (CDK)s are often drivers of cancer pathogenesis. CDK4/6 are critical mediators of cellular transition into S phase and important for cancer initiation, growth, and survival. Previously we reported a novel brain penetrant CDK4/6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/6 and >2000-fold selectivity against other CDKs. PRT3645 inhibits cellular phosphorylation of Rb with nanomolar potency. Furthermore, PRT3645 exhibits favorable in-vivo safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog and non-human primates. Here, we explored the therapeutic potential of PRT3645 in tumor models harboring actionable genomic alterations but lacking effective targeted treatments. Specifically, we explored the combination of PRT3645 with a selective estrogen receptor degrader (SERD), an approved treatment for patients with HR+/HER2-, ESR1-mutated advanced or metastatic breast cancer. In an ESR1 mutated breast cancer PDX model, PRT3645 not only exhibited monotherapy activity but also induced significant tumor regression in combination with a SERD through inhibiting ER-mediated signaling. We also investigated PRT3645 single agent activity and combination with a clinically validated MEK inhibitor in a BRAF class III mutant colorectal CDX model. PRT3645 was well-tolerated and demonstrated significant anti-tumor efficacy, which was further enhanced by combination therapy. To further explore comprehensive inhibition of cell cycle progression and overcome potential resistance to CDK4/6 inhibition, we investigated dual inhibition of CDK4/6 and CDK2. CDK2 is a crucial player in regulating the cell cycle by controlling the late G1/S transition, promoting DNA replication, and contributing to the DNA repair processes. Additionally, CDK2 activation has been identified as a potential mechanism of resistance to CDK4/6 inhibition. We evaluated CDK2 and CDK4/6 dual inhibition in CDKN2A loss or CCNE amplified cancer cell lines and observed enhanced proliferation suppression, cell cycle arrest and senescence induction, underscoring the potential of this combination for improved therapeutic efficacy across various cancer types. In summary, PRT3645 displays an excellent balance of potency, selectivity, pharmacokinetic parameters across species as well as brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with other targeted therapies, offering potential benefits across a wide spectrum of cancer types. In addition, the concept of dual inhibition of CDK4/6 and CDK2 presents a rational and promising approach to enhance the efficacy of cancer therapy. Citation Format: Yue Zou, Kirsten Gallagher, Srijita Dhar, Andrew Busking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Andrew Combs, Kris Vaddi, Sandy Geeganage, Peggy Scherle, Sang Hyun Lee. The brain penetrant CDK4/6 inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of breast cancer, CRC and NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5710.

Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of <300 nM. PRT3645 exhibits favorable in-vitro safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog, and non-human primates. In addition to robust monotherapy activity observed in preclinical models of ER+/HER2- breast cancer, we explored the activity of PRT3645 in other tumor types as well as in combination with other targeted therapies. In NSCLC, PRT3645 treatment resulted in significant inhibition of cell lines that harbor activation of the RAS/MEK/ERK pathway in proliferation assays and demonstrated comparable high synergy scores when combined with clinically approved covalent KRAS G12C inhibitors. In-vivo, oral PRT3645 was well tolerated and induced anti-tumor efficacy in two KRAS G12C mutant xenograft models that harbor the CDKN2A (p16) deletion. Anti-tumor efficacy was further improved when PRT3645 was combined with KRAS/MEK inhibitors in xenograft models and the combination therapy was well tolerated. In addition, we explored combinations of PRT3645 with a brain penetrant receptor tyrosine kinase inhibitor (TKI), an approved treatment for patients with advanced HER2+ breast cancer, including patients with brain metastases. In a HER2+ orthotopic human breast cancer brain metastasis model, PRT3645 was highly efficacious in combination with HER2 kinase inhibition and enhanced median survival significantly. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species and brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with KRAS/MEK inhibitors, and with a brain penetrant HER2 receptor TKI, both in-vitro and in-vivo. PRT3645 has advanced into Phase 1 clinical trials (NCT05538572). Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2300.

Abstract MCL1 is a member of the anti-apoptotic BCL2 family of proteins and plays a critical role in maintaining cellular homeostasis and promoting cell survival. It is frequently amplified in cancer and increased expression of MCL1 is associated with a higher grade and poor prognosis in multiple tumor types. Importantly, MCL1 has been implicated in mediating resistance to chemotherapy as well as targeted therapies, including the BCL2 inhibitor, venetoclax. Here, we describe the in vitro and in vivo activity of PRT1419, a potent and selective inhibitor of human MCL1, that can induce tumor cell death by apoptosis. PRT1419 inhibits the binding of MCL1 to its physiological ligand, BIM, with low nanomolar potency. PRT1419 also demonstrated >200-fold selectivity against other BCL2 family members, including BCL2 and BCL-XL. In vitro, PRT1419 treatment resulted in robust activation of apoptotic markers such as cleaved caspase-3 in a concentration-dependent manner in several cancer cell lines. Consistent with its pro-apoptotic effects, PRT1419 treatment led to robust inhibition of cell proliferation in a concentration-dependent manner in a panel of cancer cell lines. Cell lines representing hematologic cancers as well as a subset of breast and non-small cell lung cancer lines were sensitive to PRT1419, and this response was associated with a significantly higher MCL1/BCL-XL mRNA ratio. Also, PRT1419 treatment resulted in potent, concentration-dependent cytotoxic activity ex vivo in patient-derived xenograft (PDX) models of various subtypes of human sarcoma, breast and esophageal cancer. PRT1419 demonstrated good oral bioavailability and favorable pharmacokinetic properties in vivo. In subcutaneous cell-line derived xenograft (CDX) models of multiple myeloma, acute myeloid leukemia (AML) and diffuse large B-cell lymphoma, oral administration of PRT1419 demonstrated potent anti-tumor activity with complete tumor regressions observed at tolerable doses. This response correlated with a dose-dependent induction of cleaved caspase-3 and cleaved-PARP in tumor tissue. Significant in vivo activity, including complete responses, was also observed in PDX models of lymphoma. In preclinical models of solid tumors, PRT1419 demonstrated significant tumor growth inhibition in a PDX model of human soft tissue sarcoma and a CDX model of breast cancer. PRT1419 was also tested in combination with other approved targeted therapies in vitro and in vivo. In AML, combining PRT1419 with a BCL2 inhibitor revealed a synergistic interaction in cell lines, ex vivo PDX models as well as a CDX model in vivo. Further, PRT1419 demonstrated synergistic activity with tyrosine kinase inhibitors to inhibit the proliferation of breast, melanoma, and non-small cell lung cancer cell lines. PRT1419 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT04543305). Citation Format: Neha Bhagwat, Alexander Grego, William Gowen-MacDonald, Min Wang, Miles Cowart, Xiaowei Wu, Jincong Zhuo, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT1419, a potent, selective and orally available inhibitor of MCL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 983.