Intrinsic and acquired resistance to CDK4/6 inhibitors (CDK4/6i) is a critical challenge in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). CDK2 inhibitors (CDK2i) show promise in this context, with several currently under early-phase clinical evaluation. Here, by analyzing tumor and liquid biopsies from patients treated with the selective CDK2i PF-07104091, we observed that PF-07104091 monotherapy achieved disease stabilization in a cohort of CDK4/6i-resistant HR+/HER2- MBC patients. Notably, responses were observed irrespective of RB expression and phosphorylation, whereas non-progressive disease appeared more frequently among patients whose tumors retained wild-type TP53. Studies in established and patient-derived cell lines further substantiated that the growth suppressive effects of CDK2i were independent of RB status in CDK4/6i-resistant models. CDK2i activity reduced DNA replication rate, increased DNA damage, and inhibited mitotic entry, with growth inhibition dependent on p53 expression. These findings indicate that CDK dependency shifts from CDK4/6 toward CDK2 as cells transition from CDK4/6i-sensitive to CDK4/6i-resistant state. The distinct mechanisms of CDK2i and CDK4/6i support enhanced synergistic activity in HR+/HER2- MBC with acquired resistance to CDK4/6i therapy.

CDK4/6 inhibitors have revolutionized treatment of hormone receptor positive (HR+), HER2 non-amplified (HER2-) breast cancer. Yet, all "dual" CDK4/6 inhibitors show common dose-limiting hematologic toxicities, foremost neutropenia. This poses challenges to provide these agents at concentrations necessary to extinguish cell cycling in tumors. HR+ breast cancer cells are highly dependent on CDK4 but not CDK6. By contrast, CDK4 is dispensable for human bone marrow derived cells, due to the primary and compensatory role of CDK6 in hematopoiesis. This prompted us to develop atirmociclib (PF-07220060), a next-generation CDK4 selective inhibitor. Atirmociclib's impact on circulating neutrophils was reduced, in proportion with its increase in CDK4 versus CDK6 selectivity. Realized dose intensification led to greater CDK4 inhibition and deeper anti-tumor responses, pointing to CDK4 target coverage as a limiting factor of CDK4/6 inhibitor efficacy. We also highlight combinatorial agents that may counter acquired resistance to CDK4 selective inhibition and widen its clinical application.

To better understand drug resistance mechanisms to CDK4/6 inhibitors and inform precision medicine, we analyze real-world multi-omics data from 400 HR+/HER2- metastatic breast cancer patients treated with CDK4/6 inhibitors plus endocrine therapies, including 200 pre-treatment and 227 post-progression samples. The prevalences of ESR1 and RB1 alterations significantly increase in post-progression samples. Integrative clustering analysis identifies three subgroups harboring different resistance mechanisms: ER driven, ER co-driven and ER independent. The ER independent subgroup, growing from 5% pre-treatment to 21% post-progression, is characterized by down-regulated estrogen signaling and enrichment of resistance markers including TP53 mutations, CCNE1 over-expression and Her2/Basal subtypes. Trajectory inference analyses identify a pseudotime variable strongly correlated with ER independence and disease progression; and revealed bifurcated evolutionary trajectories for ER-independent vs. ER-dependent drug resistance mechanisms. Machine learning models predict therapeutic dependency on ESR1 and CDK4 among ER-dependent tumors and CDK2 dependency among ER-independent tumors, confirmed by experimental validation.

A dysregulated cell cycle is a hallmark of cancer and inhibition of cyclin-dependent kinases (CDK) is a proven therapeutic strategy in treating hormone receptor-positive/HER2- breast cancer and a variety of other cancers. 18F-3'-deoxy-3'-fluorothymidine (18F-FLT) is a validated PET biomarker to measure cell proliferation. In this study, we show the utility of 18F-FLT PET imaging as a pharmcodynamic biomarker in differentiating the efficacy of PF-07104091 (CDK2-selective inhibitor) in palbociclib (CDK4/6 inhibitor)-sensitive and -resistant tumor models. 18F-FLT PET imaging was performed after 4 days of treatment with CDK inhibitors and IHC biomarkers of tumor cell proliferation (Ki67 and pRb) were evaluated for correlation. Tumor growth inhibition studies demonstrated that palbociclib was efficacious in an MCF7 model but not in an OVCAR-3 model, whereas PF-07104091 showed dose-dependent tumor growth inhibition in both MCF7 and OVCAR-3 models. Consistent with this observation, 18F-FLT PET was able to differentiate the resistance to palbociclib from sensitivity to PF-06873600 (CDK2/4/6 inhibitor) and PF-07104091 in the OVCAR-3 model. In contrast, the 18F-FLT PET biomarker showed reduced uptake in the MCF7 model after treatment with both palbociclib and PF-07104091. Similarly, PF-07104091 demonstrated reduced 18F-FLT uptake in NIBR-5493, an ovarian cancer patient-derived xenograft model. IHC biomarkers Ki67 and pRb correlated with the 18F-FLT uptake trends in all three tumor models. This work highlights the utility of 18F-FLT PET as a quantitative, noninvasive biomarker which provides whole-body information. 18F-FLT PET has potential to be a biomarker in novel CDK inhibitor clinical trials to evaluate palbociclib resistance and to identify responding and nonresponding patients.

By virtue of its role in cellular proliferation, microtubule-associated serine/threonine kinase-like (MASTL) represents a novel target and a first-in-class (FIC) opportunity to provide a new impactful therapeutic agent to oncology patients. Herein, we describe a hit-to-lead optimization effort that resulted in the delivery of two highly selective MASTL inhibitors. Key strategies leveraged to enable this work included structure-based drug design (SBDD), analysis of lipophilic efficiency (LipE) and novel synthesis. The resulting advanced lead compounds enabled a tumor growth inhibition study which was pivotal in assessing the potential value of MASTL as an oncology therapeutic target.

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

More than half of the similar to 20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile-cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

Abstract Cell cycle/tumor repressors can be pharmacologically activated to repress uncontrollable proliferation of cancer cells. However, targeting cell cycle regulators of G2/M and mitotic checkpoints to achieve maximum therapeutic benefit remains a challenge due to a limited therapeutic index. Here, we identified highly potent and selective oral bioavailable clinical candidate inhibitors to the first-in-class oncology target Microtubule Associated Serine/Threonine Kinase-Like (MASTL) aimed at expanding therapeutic control of the cell cycle for cancer therapy. Breath of efficacy screening in multiple tumor indications in vitro followed by computational analyses identified the regulatory subunit PPP2R2A as a potential patient selection biomarker that strongly correlates to MASTL inhibitor response irrespective of cancer genetic alterations. Functional genetics studies confirmed that sensitivity to MASTL inhibition is defined by PPP2R2A expression. To identify genetic mediators of MASTL inhibitor resistance, a whole genome CRISPR knockout screen with the MASTL inhibitor was performed and further established that the MASTL inhibitor response is dependent on PPP2R2A containing PP2A complexes. Mechanistic studies (proteomics, resistance, functional genetics, and time-lapse microscopy) demonstrated that MASTL inhibition disrupts mitosis by activating the tumor suppressor PP2A, leading to a switch from CDK1 activity to CDK1 inactivity, mitotic defects, and cancer cell death. MASTL inhibition treatment in tumor models in vivo and in patient-derived organoid models results in potent tumor growth inhibition. Our results demonstrate that the first identified highly selective MASTL clinical candidate inhibitors represent a potential new cancer therapeutic strategy to target mitosis and mitotic exit by activating the tumor repressor PP2A to counter CDK1 activity. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process. Citation Format: Chad M. Toledo, Koleen J. Eisele, Dong Hyun Kim, Xinmeng Mu, Sonja Brun, Carl Davis, Eric C. Greenwald, Fion Hui, Meilan He, Yanling Yang, Ana Flores-Bojorquez, Yuxin Li, Penney L. Khamphavong, Joseph H. Lee, Jian Li, Dana J. Ramms, Greg Jones, Marin Auth, Cory L. Painter, Andrew Nager, Jon Oyer, Sergei L. Timofeevski, William E. Pierceall, Anwar Murtaza, Jonathan R. Heyen, Rebecca Gallego, Casey Quinlan, Indrawan McAlpine, Todd L. VanArsdale. MASTL small-molecule inhibition to target mitosis for cancer therapy [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 5903.

Abstract PF-07220060 is a selective inhibitor of CDK4, displaying ~20-fold and ~4-fold increased selectivity for CDK4 versus CDK6 when compared to palbociclib and abemaciclib/ribociclib, respectively. This translates to less PF-07220060 associated neutropenia in beagle dogs and humans when compared to dual CDK4/6 inhibitors. Because neutropenia is the primary culprit necessitating limited and often interrupted clinical dosing of dual CDK4/6 inhibitors, the advantage of PF-07220060 lies in the flexibility to escalate the drug’s exposure in patients and thus realize near-complete target coverage of the CDK4 oncogene in tumors that are dependent on this kinase. Here we select tumor types/indications that show dependency on CDK4 but not CDK6 and includes cells derived from luminal breast cancer and androgen-receptor positive metastatic prostate cancer. Additionally, we investigate certain drugs with which PF-07220060 may be combined to maximize its efficacy in these tumor indications. PF-07220060 sensitizes HR+ HER2- breast cancer to the estrogen inhibitor, fulvestrant and the degrader, ARV-471 (vepdegestrant). PF-07220060 + vepdegestrant showed significantly longer tumor regrowth delay vs monotherapy groups and vs Palbo + vepdegestrant and vs PF-07220060 + fulvestrant*. Similarly, PF-07220060 sensitizes prostate cancer to the androgen receptor antagonist, enzalutamide. In HR+ HER2- breast cancer cells, the senescent cell fraction was markedly increased by co-treatment with PF-07220060 and fulvestrant versus either drug alone, albeit the combination did not trigger tumor cell death. Further addition of the PI3K inhibitor alpelisib was sufficient to enforce tumor shrinkage in vivo. Alternatively, inhibition of CDK4 plus CDK2 also led to tumor shrinkage in xenograft models of HR+, HER2- breast cancer. Finally, our preclinical data indicate that the propensity of CDK6 to compensate for CDK4 inhibition in these tumor types is limited. Compensation by CDK6 was seen in only a fraction of the evaluated in vitro and in vivo models. Even so, in these instances, PF-07220060’s efficacy remained comparable to palbociclib’s when both drugs were used at their therapeutic doses. We conclude that PF-07220060’s anti-tumor efficacy is broadly superior to currently approved dual CDK4/6 inhibitors. Citation Format: Lars Anders, Bernadette Pascual, Britton Boras, Julie Cianfrogna, Scott Garza, Na Li, Jing Tang Yuan, Mark Moen, Nanni Huser, Gary Gallego, Mehran Jalaie, Sacha Ninkovic, Sujin Cho-Schultz, Hong Shen, John Kath, Klaus Dress, Wade Diehl, Sajiv Nair, Rhys Jones, Jennifer Lafontaine, Anwar Murtaza, Aida Sacaan, Sudhakar Chintharlapalli, Todd VanArsdale. Preclinical development of the CDK4 selective inhibitor PF-07220060: Increased CDK4 versus CDK6 inhibition leads to improved anti-tumor efficacy at therapeutic concentrations [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 595.

Abstract The selective inhibition of Cyclin\CDK complexes controlling cell cycle progression has been established as cancer therapy by selective CDK4/6 inhibitors in HR+/HER2- breast cancer. Expanding control of the cell cycle through selective inhibition of CDK2 offers novel therapeutic opportunities in cancer, including targeting CCNE1 amplified tumors and countering resistance to CDK4\6 inhibitors in ER+ breast cancer. PF-07104091 is a first-in-class CDK2-selective inhibitor under clinical investigation in patients with HR+/HER2- breast cancer and ovarian cancer. Preclinical studies using PF-07104091 establish the therapeutic impact of CDK2 inhibition in both diseases and highlight distinct mechanistic roles for CDK2 in control of cancer cell proliferation. In models of CCNE1 amplified ovarian cancer, CDK2 plays the dominant role in control of RB1 phosphorylation and the G1 checkpoint. CDK2 inhibition with PF-07104091 induces G1 growth arrest and controls tumor xenograft growth as single agent therapy. In ER+ breast models CDK2 plays a supportive role in the control of RB1 phosphorylation cooperating with CDK4\6. Whole genome CRISPR KO and CRISPR activation screens in conjunction with CDK4\6 inhibition establish CDK2 KO as a primary sensitizer to CDK4\6 inhibition, and support Cyclin E\CDK2 complexes as the driver of resistance to CDK4\6 inhibitors in ER+ breast models. PF-07104091 combined with CDK4\6 inhibitor Palbociclib or CDK4-selective inhibitor PF-0060 synergistically controls proliferation of ER+ BC cells in vitro and induces tumor regression in ER+ BC xenograft models, including PDX models with acquired resistance to CDK4\6 inhibitors and endocrine therapy. Citation Format: Chen Shen, Ming Qiu, Nanni Huser, Bernadette Pascual, Qin Zhang, Todd VanArsdale, Anwar Murtaza, Jonathan Almaden, berly Kim, Lars Anders, Koleen Eisele. PF-07104091, a first-in-class CDK2-selective inhibitor for the treatment of HR+/HER2- breast cancer and CCNE1high ovarian cancer [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 5709.

Supplemental figure 5: IHC analysis of treated PDX tumors indicated on-target activity of bazedoxifene and palbociclib

Supplemental figure 3: SERDs and palbociclib inhibit breast cancer cell proliferation by independent mechanisms

PDF file - 231 KB, Schematic representation of CRC GEMM conditional alleles

Supplementary Figure S1. Anti-4-1BB and anti-PD-1 synergized to suppress B16F10 tumor growth. Supplementary Figure S2. The flow cytometric gating strategy to analyze immune cells. Supplementary Figure S3. Elevated expression of genes associated with anti-tumor immune response in MC38 colon carcinomas. Supplementary Figure S4. Accumulation of CD8+ T cell and memory phenotype differentiation in MC38 model. Supplementary Figure S5. Serum liver enzyme levels and hematology alterations in response to 4-1BB activation therapy in B16F10 tumor-bearing mice.

PDF file - 1 MB, Intrasplenic injection of GEMM tumor derived cell lines and tumor growth in the liver

Supplementary Figures 1 - 5 from PF-03732010: A Fully Human Monoclonal Antibody against P-Cadherin with Antitumor and Antimetastatic Activity

Supplemental figure 2: SERDs inhibit the constitutive activity of the clinically relevant Y537N,D538G ESR1 double mutant

Supplementary Figure Legends

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

XLS file - 25 KB, Top 5 Ingenuity Pathway Analysis biological functions represented in GEMM AP vs AKP signature